The human cytomegalovirus (HCMV) is a significant public health concern in the United States. Most important are the effects of the virus on developing fetuses and immunocompromised individuals where it causes a variety of pathological conditions ranging in severity from mild to life-threatening. Since HCMV is present in a persistent or latent form in 50-90% of the world's adult population, the identification of virl gene products that contribute to viral trafficking, persistence, and horizontal transmission is an intense and important area of investigation. Interestingly, HCMV encodes 4 genes that are homologous to cellular G- protein coupled receptors (GPCRs). The HCMV GPCRs are not essential for viral replication in vitro, however their homology to cellular GPCRs suggests that they may profoundly affect cellular physiology to ensure replication of the viruses in organs important for pathogenesis. The strict species specificity of HCMV has precluded an analysis of the function of HCMV GPCRs in vivo. However, as the biology of the related murine cytomegalovirus (MCMV) is similar to that of HCMV, the murine virus has served as a useful model for studying how the vGPCRs affect cytomegalovirus pathogenesis in vivo. Interestingly, the MCMV encoded M33 GPCR is essential for replication within the salivary gland of infected mice, suggesting that M33 activity may have a direct impact on viral persistence and/or horizontal transmission of virus. Based on our preliminary data, we hypothesize that M33 signaling through the cellular G?q/G?11 pathway alters salivary acinar cell physiology leading to amplification and spread of the virus within the salivary epithelium. The proposed studies are highly significant as the salivary gland and salivary secretions play an important role in horizontal transmission of cytomegaloviruses, yet little is known about the viral properties that facilitate viral amplification within the gland and promote movement of virus into the saliva. In aim 1, we will use knockout, transgenic and pharmacological approaches to test the hypothesis that G?q/G?11 is the proximal signaling pathway used by M33 for MCMV growth within the salivary gland in vivo. In aim 2 we will test whether M33 altered acinar cell viability or secretor activity facilitates amplification and/or spread of virus in the gland. In aim 3 we will use novel primary salivary gland derived salisphere approaches to study signaling via MCMV and HCMV GPCRs in salivary cells and also use this approach to investigate the requirement for HCMV GPCRs for viral growth in salivary gland implants in vivo. The innovative experiments proposed in this application will lead to novel insights into the function of cytomegalovirus GPCRs and into mechanisms by which cytomegaloviruses persist and gain access to fluids important for horizontal transmission. Defining essential roles for cytomegalovirus GPCRs in promoting salivary gland replication and spread could ultimately lead to the development of unique antivirals designed to prevent cytomegalovirus transmission via saliva.